The updated relationship between the cleft‑lip and palate transmembrane protein‑1‑like rs401681 and lung cancer risk: A systematic review and meta‑analysis

Currently, the role of cleft-lip and palate transmembrane protein-1-like (CLPTM1L) rs401681 in various tumor types, particularly lung cancer, has garnered significant attention. However, the findings across studies have shown discrepancies. The aim of the present meta-analysis was to provide a more nuanced understanding of the involvement of CLPTM1L rs401681 in lung cancer development. Several electronic databases were systematically searched, including PubMed, Cochrane Library, Embase, Medline, Wanfang, Google Scholar and Chinese National Knowledge Infrastructure. Odds ratios (ORs) and 95% confidence intervals (CIs) were synthesized using random-effects models. Heterogeneity of included studies was assessed using the I2 statistic and Q test. Sensitivity analysis was conducted to evaluate the stability of overall estimates. Moreover, Egger's test was utilized to detect potential publication bias. The collective ORs indicated a significant association between the CLPTM1L rs401681 polymorphism and susceptibility to lung cancer across various genetic comparisons. These encompass allele T vs. allele C (OR=0.93, 95% CI=0.88-0.99, P<0.001), TT + CT vs. CC (OR=0.91, 95% CI=0.87-0.96, P<0.001), TT vs. CC + CT (OR=0.88, 95% CI=0.80-0.96, P<0.001), TT vs. CC (OR=0.84, 95% CI=0.75-0.94, P<0.001) and CT vs. CC (OR=0.84, 95% CI=0.75-0.94, P<0.001). Examination through statistical Q test and I2 statistic revealed pronounced heterogeneity across four genetic comparisons (allele T vs. allele C, TT + CT vs. CC, TT vs. CC and CT vs. CC). Ethnical distinctions emerged as the primary, if not exclusive, sources of the significant heterogeneity. Upon stratification by ethnicity, a notable reduction in heterogeneity was discernible within the Caucasian demographic. However, heterogeneity persisted within the Asian population. Furthermore, lung cancer risks were statistically significantly decreased for individuals possessing allele T through all genetic comparisons within Caucasians; whereas among Asians, significant reduction was observed solely in the TT vs. CC comparison. The present meta-analysis uncovers a significant association between the CLPTM1L rs401681 polymorphism and altered susceptibility to lung cancer.


Introduction
Lung cancer currently ranks among the foremost causes of cancer death, with its incidence and fatality rates escalating annually (1).In 2012, ~1.8 million new cases of lung cancer were reported globally, resulting in 1.6 million fatalities (1).Epidemiological data suggest an estimated 230,000 new cases of lung cancer in the USA in 2022.Furthermore, the mortality rate attributed to lung cancer is anticipated to surpass the combined rates of breast, prostate and colon cancer (2).These conditions impose substantial economic and psychological burdens on society and families.While smoking is a well-established risk factor for lung cancer, accounting for 80-90% of the cases, it is noteworthy that only 15% of smokers ultimately develop lung cancer.This suggests that hereditary predisposition might also significantly contribute to lung cancer (1,3).Histologically, lung cancer is broadly classified into small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) (4).A total of ~80-85% lung cancers are NSCLC, encompassing various subtypes such as squamous cell carcinoma, adenocarcinoma, large cell carcinoma, adeno-squamous carcinoma and sarcomatoid carcinoma (4).Multiple genetic alterations have shown the potential to disrupt biological process or functionals, thereby facilitating the onset of lung cancer.Hence, the identification of susceptibility genes for lung cancer holds paramount clinical significance.
Cleft-lip and palate transmembrane protein-1-like (CLPTM1L) protein is identified as a protein associated with cisplatin resistance (5,6).The gene is situated on chromosome 5p15.33 and encodes a protein comprising 538 amino acids (6) with a molecular weight of 62 KDa (3,7).Functionally, the protein encoded by CLPTM1L is a membrane entity implicated in cisplatin resistance, where its overexpression in cisplatin-sensitive cells triggers apoptosis (8).Remarkably, both human and murine proteins exhibit 99% sequence identity, featuring 538 amino acids (5).Notably, within the chromosomal region 5p15, housing two genes, telomerase reverse transcriptase (TERT) and CLPTM1L, a susceptibility locus for lung cancer has been identified, with a potential role attributed to NKX2.4 (9).Nonetheless, the interaction mechanism between these genes remains elusive.Moreover, CLPTM1L has been implicated in modulating inflammatory responses (10).Given the profound link between malignancy and heightened inflammation (11), numerous studies have scrutinized the potential of CLPTM1L mutation in precipitating tumorigenesis.Genome-wide association studies (GWAS) have pinpointed specific single nucleotide polymorphisms (SNPs) within the CLPTM1L gene (such as rs401681 and rs402710) as intricately intertwined with the onset and progression of various malignancies spanning lung cancer, breast cancer, pancreatic cancer, nasopharyngeal carcinoma and bladder cancer among others (3,7,12).In addition, clinical analyses have revealed that the expression levels of CLPTM1L in lung cancer tissue surpass those in healthy lung tissue, particularly evident in adenocarcinoma cells (5).Adenocarcinoma cells exhibit an average 2.15-fold elevation in CLPTM1L expression over normal immortalized cell lines (P = 3.59x10 -7 ) (13).Since its initial identification of association with lung cancer susceptibility, numerous subsequent investigations have delved into the relationship between lung cancer risk and the rs401681 variant.Nevertheless, subsequent studies have yielded disparate findings (14)(15)(16)(17)(18)(19)(20).To comprehensively elucidate the association between lung cancer and rs401681, a meta-analysis encompassing correlational studies published prior to 2023 was undertaken.
Selection criteria.The inclusion criteria for eligible studies were as follows: i) Studies comprising both control and case groups; ii) studies investigating the association between CLPTM1L rs401681 and lung cancer susceptibility; iii) studies providing adequate data on genotype frequencies, or odds ratio (OR) with 95% confidence interval (95% CI) for lung cancer risk assessment and iv) investigations involving human subjects.A study that meets any of the following criteria was deemed to be non-compliant with the present meta-analysis: i) Comments, abstracts for conference, case reports, editorials or reviews; ii) insufficient data for OR calculation; iii) animal studies; and iv) studies lacking control groups.
Quality assessment.The quality of all included studies was assessed utilizing the Newcastle-Ottawa Scale (NOS).The NOS assesses studies across three key dimensions: The selection of the study groups; the comparability of these groups; and the ascertainment of either the exposure or outcome of interest for case-control or cohort studies respectively.The scale yields a maximum score of 9 (21).Finally, the studies with a total score surpassing 6 are considered high quality, those scoring between 4 and 6 are considered medium quality, and studies with a total score below 4 are categorized as low quality.
Data extraction.Data extraction was independently carried out by two investigators.Any discordant opinion was solved through discussing to reach a final consensus.The data obtained from each included study encompassed the first author's name, publication year, region, ethnicity, disease type, sample size, genotype and/or allele frequencies of case and control subjects, and testimony of Hardy-Weinberg equilibrium in the control group.
Statistical analysis.The statistical analyses were conducted using STATA software (version 17.0, StataCorp LP).The effect size of the correlation between the CLPTM1L rs401681 polymorphism and lung cancer risk was evaluated by pooled ORs with corresponding 95% CIs.χ 2 -based Q-test and I 2 statistic were used to assess heterogeneity among studies.The random-effects model was used for the present meta-analysis.The significant heterogeneity was defined as P<0.05 for the Q test or I 2 >50%.Moreover, subgroup analysis was used to investigate potential sources of substantial heterogeneity.Publication bias was assessed by Egger's linear regression test.To test the robustness of the pooled outcomes, sensitivity analysis was employed using the leave-one-out method to identify the influence of individual studies on the overall effect estimate.P<0.05 was considered to indicate a statistically significant.
In silico analysis.Aiming to elucidate the impact of polymorphisms on CLPTM1L, the authors embarked on an exploration of the association between the rs401681 and CLPTM1L expression levels via Genotype-Tissue Expression (GTEx) Analysis Release V6 (dbGaP Accession phs000424.v7.p2) data (22).To further investigate the potential influence of CLPTM1L expression on tumorigenesis, the authors endeavored to mine evidence from The Cancer Genome Atlas (TCGA) database (https://www.cancer.gov/ccg/research/genome-sequencing/tcga) by using the Kaplan-Meier.Additionally, the recently developed interactive platform, Gene Expression Profiling Interactive Analysis, was utilized to augment the inquiry (23)(24)(25).
Sensitivity analysis.From the results of sensitivity analysis in various comparisons (Figs.S11-15), no substantial alterations in the re-obtained ORs were observed when compared with the initial ORs.This indicated the robustness and consistency of these findings.
Moreover, whether the expression level of CLPTM1 influences the disease-free survival (DFS) and overall survival (OS) of lung cancer was investigated.The Kaplan-Meier estimations revealed a notable discrepancy in OS between the low and high CLPTM1 transcripts per kilobase million (TPM) groups in LUAD (log-rank P=0.04), albeit a difference in DFS close to statistical significance (log-rank P= 0.063).On the other hand, no distinction was observed between high and low CLPTM1 TPM groups in LUSC (log-rank P=0.84 for OS and 0.27 for DFS).Details are presented in Fig. 3B and C.

Discussion
In the present study, the impact of CLPTM1L rs401681 within the locus 5p15.33 on lung cancer incidence was scrutinized, and it was found that it had a remarkable association with onset of lung cancer.Prior investigations yield a discordant landscape regarding the role of CLPTM1L rs401681 in the occurrence of lung cancer.There was no overall correlation between CLPTM1L rs401681 and the risk of lung cancer in several previous studies (20,28), whereas others reported that allele T of CLPTM1L rs401681 decreases the susceptibility of lung cancer (33,39,40,43).A precedent meta-analysis hinted at an association between CLPTM1L rs401681 and lung cancer (14), albeit with a caveat on sample size constraints acknowledged by the authors.Subsequently, emerging studies exploring the relationship between CLPTM1L rs401681 and lung cancer risk have been published (16,18,20,32,34,39,41,44,45).To further determine the relationship between CLPTM1L rs401681 polymorphism and lung cancer risk, the authors embarked on a meta-analysis of all the literature published before 2023.In the present study, the association between CLPTM1L rs401681 polymorphism and lung cancer risk was systematically reviewed and was based on ten case-control studies, encompassing 14,195 cases and 16,732 controls.The meta-analysis demonstrated that CLPTM1L rs401681 polymorphism was associated with the occurrence of lung cancer in all the genetic comparisons investigated, suggestive of a protective effect conferred by the T allele of CLPTM1L rs401681 mutation against lung cancer.Nevertheless, the I 2 statistic and statistical Q test revealed significant heterogeneity, with ethnic origin emerging as a plausible primary contributor to this observed heterogeneity.
Upon stratification by race, a statistically significant association of CLPTM1L rs401681 with lung cancer risk was observed in each genetic comparison among Caucasians, whereas it was only notable for the TT vs. CC comparison among Asians.Although the exact cause for racial disparities is not entirely clear, one possible cause may lie within the genetic context.When considering subgroups based on histology, it was found that the association between rs401681 and NSCLC or SCLC risks was not statistically significant for all comparisons.This suggested that CLPTM1L rs401681 variant genotypes may not exert an effect on the pathological  differentiation of lung cancer.Future investigations should aim to elucidate the underlying mechanisms, taking into account factors such as histology or race heterogeneity, to provide a more nuanced understanding of the impact of CLPTM1L gene polymorphism on lung cancer risk and to tailor personal therapy.
The association between lung cancer and the CLPTM1L rs401681 polymorphism may be confounded by other influential SNPs, particularly those situated within TERT-CLPTM1L region.This is especially pertinent for SNPs in close linkage disequilibrium with rs401681, such as rs31490 and rs414965, or those directly implicated in lung cancer.In addition, variations in matching criteria, selection bias, and limited data availability in the present study may lead to statistical insufficiency to identify subtle differences, and a fluctuated risk estimate.Several findings underscored the critical importance of assessing genetic influences across diverse populations for disease onset and progression.
The present study possessed several limitations.Primarily, the intricate interplay between gene-gene and gene-environment interactions in lung cancer risk poses challenges for evaluating genotype polymorphism alongside potential psychological and environmental factors.Consequently, the assessment primarily focused on delineating the risk coefficient associated with aberrant genotypes.Secondly, the present study only included Chinese and English studies, potentially introducing unacknowledged language bias.Thirdly, the possibility of chance findings cannot be entirely discounted, given the limited patient cohorts within the present stratified analyses.These subgroup associations necessitate validation within larger cohorts for enhanced precision.Lastly, due to lacking certain subgroup data of included studies, some important subgroups such as sex are not analyzed in the present meta-analysis.In future research, investigators should focus more on this important issue.
In conclusion, the current meta-analysis demonstrates a significant association between the CLPTM1L rs401681 polymorphism and the risk of lung cancer, wherein the T allele exhibits a protective effect.These findings offer preliminary insights into assessing the risk of lung cancer based on the SNP rs401681 genotype, thus laying the groundwork for personalized medicine endeavors.

Figure 1 .
Figure 1.Flowchart for literature searching and selection.
-Ottawa Scale; HWE, Hardy-Weinberg equilibrium; Y, conform to HWE.TableII.Comparative analysis of genotype frequencies and the risk of lung cancer in the subgroups.
-small cell lung cancer; SCLC, small cell lung cancer.Table III.Result of meta-analysis for CLPTM1L rs401681 polymorphism and lung cancer risk.T vs. C TT + CT vs. CC TT vs. CC + CT TT vs. CC CT vs. CC

Figure 2 .
Figure 2. In silico analysis of CLPTM1L expression concerned to rs401681 and lung cancer.CLPTM1L mRNA expression by expression quantitative trait loci analysis in human tissues based on Genotype-Tissue Expression database.CLPTM1L, cleft-lip and palate transmembrane protein-1-like.

Figure 3 .
Figure 3.In silico analysis of CLPTM1L expression.(A) Comparison of CLPTM1L expression between tumor site and matched normal tissue in The Cancer Genome Atlas database.(B) The association between the expression of CLPTM1L and the overall survival time and disease-free survival time of LUAD.(C) The association between the expression of CLPTM1L and the overall survival time, disease free survival time of LUSC.CLPTM1L, cleft-lip and palate transmembrane protein-1-like; LUAD, lung adenocarcinoma; LUSC, lung squamous cell carcinoma.*** P<0.001.